Circ_FURIN knockdown assuages Testosterone-induced human ovarian granulosa-like tumor cell disorders by sponging miR-423-5p to reduce MTM1 expression in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among reproductive-age women. The mechanism by which circular RNA (circRNA) drives PCOS development remains unclear. Thus, the study is designed to explore the role of a novel circRNA, circ_FURIN, in the PCOS cell model and the underlying mechanism. METHODS: PCOS cell model was established by treating human ovarian granulosa-like tumor cells (KGN) with Testosterone (TTR). RNA expressions of circ_FURIN, microRNA-423-5p (miR-423-5p) and myotubularin 1 (MTM1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was checked by Western blot. Cell proliferation was investigated by a 5-Ethynyl-29-deoxyuridine assay, 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis for cell cycle. Apoptotic cells were quantified by flow cytometry analysis for cell apoptosis. The interplay between miR-423-5p and circ_FURIN or MTM1 was identified by dual-luciferase reporter and RNA pull-down assays. RESULTS: Circ_FURIN and MTM1 expressions were significantly upregulated, whereas miR-423-5p was downregulated in the ovarian cortex tissues of PCOS patients and TTR-treated KGN cells compared with controls. Circ_FURIN depletion relieved TTR-induced proliferation inhibition and apoptosis promotion. Besides, knockdown of miR-423-5p, a target miRNA of circ_FURIN, rescued circ_FURIN knockdown-mediated effects under TTR treatment. MiR-423-5p remitted TTR-induced cell disorders by binding to MTM1. Moreover, circ_FURIN modulated MTM1 expression through miR-423-5p. CONCLUSION: Circ_FURIN silencing protected against TTR-induced dysfunction by the miR-423-5p/MTM1 pathway in human ovarian granulosa-like tumor cells.

Soy promotes juvenile granulosa cell tumor development in mice and in the human granulosa cell tumor-derived COV434 cell line.

Soy attracts attention for its health benefits, such as lowering cholesterol or preventing breast and colon cancer. Soybeans contain isoflavones, which act as phytoestrogens. Even though isoflavones have beneficial health effects, a role for isoflavones in the initiation and progression of diseases including cancer is becoming increasingly recognized. While data from rodent studies suggest that neonatal exposure to genistein (the predominant isoflavone in soy) disrupts normal reproductive function, its role in ovarian cancers, particularly granulosa cell tumors (GCT), is largely unknown. Our study aimed to define the contribution of a soy diet in GCT development using a genetically modified mouse model for juvenile GCTs (JGCT; Smad1 Smad5 conditional double knockout mice) as well as a human JGCT cell line (COV434). While dietary soy cannot initiate JGCT development in mice, we show that it has dramatic effects on GCT growth and tumor progression compared to a soy-free diet. Loss of Smad1 and Smad5 alters estrogen receptor alpha (Esr1) expression in granulosa cells, perhaps sensitizing the cells to the effects of genistein. In addition, we found that genistein modulates estrogen receptor expression in the human JGCT cell line and positively promotes cell growth in part by suppressing caspase-dependent apoptosis. Combined, our work suggests that dietary soy consumption has deleterious effects on GCT development.

Granulosa cell tumor of the ovary after long-term use of tamoxifen and toremifene.

The relation between the use of tamoxifen and gynecologic tumors has been documented. In this case, a 58-year-old postmenopausal woman had been treated with tamoxifen for 5 years followed by toremifene for 1.5 years due to the presence of stage II estrogen receptor-positive breast cancer. The patient was found to have a stage Ic granulosa cell tumor of the ovary despite undergoing annual gynecologic examinations. This report presents a case of granulosa cell tumor of the ovary after the long-term use of tamoxifen and toremifene.

Granulosa cell tumor of the ovary and antecedent of adjuvant tamoxifen use for breast cancer.

BACKGROUND: Adult granulosa cell tumor associated with antecedent use of tamoxifen as adjuvant hormonotherapy for breast cancer is rare. The pathogenesis of this occurrence remains difficult to explain. The estrogenic effect of tamoxifen can be one such explanation. CASE PRESENTATION: A 47 year-old women was treated with surgery, chemotherapy, radiotherapy and tamoxifen for stage III estrogen receptor positive breast carcinoma. Ten months after stopping tamoxifen, we diagnosed a stage Ic granulosa cell tumor of the ovary. CONCLUSIONS: Use of tamoxifen has been found to be associated with gynecological tumors like endometrial carcinoma. Its association with granulosa cell tumor of the ovary is uncommon. Only two previous cases have been reported in literature.

LH analog and dietary isoflavones support ovarian granulosa cell tumor development in a spontaneous mouse model.

The reproductive hormone environment is an important influence upon spontaneous ovarian granulosa cell (GC) tumor development in genetically susceptible (SWR x SWXJ-9) F1 female mice: androgenic support during puberty stimulates tumorigenesis, while exposure to 17beta-estradiol (E(2)) suppresses tumor initiation. We sought to determine whether gonadotropic stimulation was sufficient to initiate GC tumors in a grafted model system, and to determine the potential for dietary isoflavones (genistein and daidzein) as alternatives to E(2) for tumor chemoprevention in vivo. Isolated ovaries from pre-pubertal (SWR x SWXJ-9) F1 females were transferred to the kidney capsule of host mice homozygous for the hypogonadal (hpg/hpg) and severe combined immunodeficiency (scid/scid) mutations. CB17; HPG-Prkdc(scid) Gnrh1(hpg)/Bm host mice received either follicle-stimulating hormone (FSH), or a functional analog for LH human chorionic gonadotropin for 2 consecutive weeks, at which time the ovary grafts were examined for evidence of tumor initiation. LH analog administration, but not FSH, initiated GC tumorigenesis in the graft system, suggesting that the LH surge at puberty initiates GC tumor development in genetically susceptible female mice. To assess the chemopreventive potential of phytoestrogens, GC tumor frequency was compared between (SWR x SWXJ-9) F1 females reared on an isoflavone-free diet versus a diet supplemented with 125 mug/g each of the isoflavones daidzein and genistein. It was observed that (SWR x SWXJ-9) F1 females reared on isoflavone-supplemented diet maintained significantly higher GC tumor frequency (22%) than females reared on isoflavone-free diet (11%), and that non-tumor-bearing siblings reared on the isoflavones had significantly increased ovarian weight, indicative of an overall stimulation of the reproductive hormone axis. The stimulation of GC tumorigenesis by isoflavones, which contrasts with the chemopreventive action of E(2) (2.5 mg/kg) administration during pubertal maturation, may result from general stimulation of ovarian growth, and the inability of the genistein and daidzein supplements to suppress LH secretion.

Proliferative lesions of ovarian granulosa cells and reversible hormonal changes induced in rats by a selective estrogen receptor modulator.

This study assessed the effects of raloxifene, a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats pergroup at the end of the treatment period and aftereach withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes. including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

Proliferative lesions of ovarian granulosa cells and reversible hormonal changes induced in rats by a selective estrogen receptor modulator.

This study assessed the effects of raloxifene. a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats per group at the end of the treatment period and after each withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes, including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

Luteinizing hormone induction of ovarian tumors: oligogenic differences between mouse strains dictates tumor disposition.

The use of fertility drugs has continued to grow since their introduction in the 1960s. Accompanying this increase has been the speculation that repetitive use of these drugs can cause ovarian tumors or cancer. We recently reported that transgenic mice with chronically elevated luteinizing hormone (LH), an analog of which is commonly used in fertility regimens, develop granulosa cell (GC) tumors. In this report we show that LH induction of these tumors is highly dependent on genetic background. In CF-1 mice, chronically elevated LH invariably causes GC tumors by 5 months of age. However, in hybrid mice generated by crossing CF-1 males with C57BL/6, SJL, or CD-1 females, elevated levels of this same hormone cause a completely different phenotype resembling a luteoma of pregnancy. We also show that three genes likely control these alternative hormonal responses. This clinical correlate of elevated LH reveals remarkably distinct, strain-dependent, ovarian phenotypes. In addition, these results support the rare incidence of GC tumors in the human population, and suggest that the ability of certain fertility drugs to cause ovarian tumors may depend on an individual's genetic predisposition.

Intrasplenic grafting of ovarian tissue containing 7, 12-dimethylbenz[a]anthracene.

To produce malignant granulosa cell tumors, ovarian tissue containing 7,12-dimethylbenz[a]anthracene (DMBA) was autografted in the spleen of 20 castrated rats. Adenocarcinoma developed in 8 rats within 6 months from the transplantation. Granulosa cell tumor arose from the intrasplenic graft, in 6 rats at the 14th month. The neoplastic cells were diffusely arranged in sheet showing focal luteinization. In 3 cases extrasplenic spreads were found on the peritoneal surface. Since malignant ovarian tumors seldom develop by intrasplenic grafting alone, the DMBA treatment may contribute to the occurrence of malignant granulosa cell tumor.

Granulosa cell tumor of the ovary associated with antecedent tamoxifen use.

BACKGROUND: Increased attention has been focused recently on the estrogenic effects of tamoxifen. Review of the literature reveals an association between tamoxifen use and gynecologic tumors. CASE: A 52-year-old postmenopausal woman was treated with tamoxifen for stage II estrogen receptor-positive breast carcinoma. Her aspartate transaminase and alanine transaminase levels increase markedly after 6 months of tamoxifen use. After an additional 17 months of elevated serum transaminases, the patient was found to have a stage Ic granulosa cell tumor of the ovary. CONCLUSION: Patients with tamoxifen-induced liver dysfunction may be at increased risk for granulosa cell tumors because of alterations in tamoxifen metabolism.

Genetic susceptibility for C19 androgen induction of ovarian granulosa cell tumorigenesis in SWXJ strains of mice.

Susceptibility to pubertal onset, malignant granulosa cell (GC) tumors of the ovary is inherited in SWR/Bm and certain SWR-related SWXJ recombinant inbred strains of mice. In some SWXJ strains, GC tumors occur spontaneously (spontaneous strains), and in others GC tumors can only be induced by treatment with dehydroepiandrosterone (DHEA-dependent strains). A gene controlling susceptibility to both spontaneous and DHEA-induced GC tumorigenesis, Gct, has been assigned to Chromosome 4. Additional research on the role of steroids in GC tumorigenesis has revealed a second gene controlling response to C19 androgenic steroids. Spontaneous strains showed increased tumor frequency after treatment with testosterone (T), whereas DHEA-dependent strains showed no GC tumors following T treatment. Within treatment groups, serum steroid data from DHEA, T, and control treated mice showed no consistent differences between spontaneous and DHEA-dependent strains with respect to progesterone, DHEA, androstenedione, dihydrotestosterone, T, estrone, or estradiol. Thus, observed differences in GC tumor responsiveness to exogenous steroids were not due to different patterns of steroid metabolism among spontaneous and DHEA-dependent strains. Further studies on the range of effective C19 steroids were conducted using one spontaneous and one DHEA-dependent strain. The spontaneous strain showed increased GC tumor frequency in response to dihydrotestosterone and androsterone treatment, whereas the DHEA-dependent strain showed no response. This result suggests that spontaneous strains may be sensitive to a broad range of C19 steroids. To determine whether genetic differences in endogenous steroid levels have a role in spontaneous GC tumorigenesis, serum steroid levels were measured in SWR/Bm and SJL/Bm progenitor strains during the developmental period of risk between 22 and 38 days of age. With the exception of transiently increased DHEA at 22 days, there were no consistent differences in steroid levels analyzed. Thus, serum steroid profiles were not reliably prognostic for GC tumorigenesis. In conclusion, GC tumor induction in response to T treatment has co-segregated with susceptibility to spontaneous GC tumors in the SWXJ recombinant inbred strains. Thus, the second gene in our ovarian granulosa cell tumor model regulates responsiveness to T. We propose to name this gene spontaneous ovarian tumorigenesis (Sot), with alleles for susceptibility (s) carried by spontaneous strains and resistance (r) carried by DHEA-dependent strains.

Neoplastic lesions induced by 1,3-butadiene in B6C3F1 mice.

1,3-butadiene (CAS No. 106-99-0) was evaluated for carcinogenicity and chronic toxicity by inhalation exposure in B6C3F1 mice because of its high production volume, widespread exposure of workers, and the lack of carcinogenicity and toxicity data (NTP Report #228, 1984). Butadiene (BD) had long been considered to have low toxicity. The long-term studies established that BD is a potent mouse carcinogen with multiple organ carcinogenicity (Huff et al. 1985; Melnick et al. 1988). This paper presents morphological descriptions and illustrations of the neoplastic lesions induced by 1,3-butadiene in B6C3F1 mice.

Induction of ovarian granulosa cell tumors in SWXJ-9 mice with dehydroepiandrosterone.

Spontaneous ovarian granulosa cell (GC) tumors develop in SWXJ-9 inbred mice at approximately the time of puberty. The effect of dehydroepiandrosterone (DHEA), a steroid secreted by the adrenals and reported to have antitumor actions, was examined in this ovarian tumor model. In contrast with expectations, administration of diet supplemented with 0.4% DHEA or Silastic capsules containing 10 mg DHEA resulted in a significant multifold increase in GC tumor incidence. Similar studies with metabolites of DHEA, i.e., testosterone (TESTO), dihydrotestosterone (DHT), and 17 beta-estradiol (E2), revealed that TESTO was as effective as DHEA in increasing GC tumor incidence. DHT was without effect, and E2 suppressed GC tumor incidence. Serum steroid levels and steroid target tissue responses were assessed to determine if a correlation between a change in level or response to specific steroids and GC tumorigenesis existed. In both tumor-free and GC tumor host mice, dietary or capsular treatment with DHEA, TESTO, or DHT resulted in substantial alteration in one or more of serum steroids, DHEA, androstenedione, TESTO, and DHT, in addition to the administered steroid. No consistent correlation was observed between changes in a single steroid or pattern of steroids and GC tumorigenesis. Although significant increases in serum estrogens could be detected in GC tumor hosts treated with DHEA but not TESTO, estrogens did not induce these tumors. Treatment with E2 increased only serum E2 levels. In tumor-free mice, DHEA and E2 treatments were associated with vaginal cytological evidence of estrogen action, whereas the androgens induced a leukocytic pattern. Eighty-eight % of GC tumor host mice, regardless of steroid treatment, showed a vaginal cytology pattern that included cornified cells. The evidence presented in this report leads us to hypothesize that (a) spontaneous and steroid-induced GC tumorigenesis in these mice have the same mechanism, and (b) subtle increases in DHEA or a closely related metabolite during the peripubertal period may initiate GC tumors in these genetically susceptible mice. The mechanism whereby these steroids initiate GC tumorigenesis remains to be determined.

Rapid induction of ovarian granulosa cell tumors by 7,12-dimethylbenz(a)anthracene in neonatally estrogenized mice.

Groups of female C3H/HeMs x 129/J F1 mice were given injections of either 20 micrograms of 17 beta-estradiol or sesame oil (vehicle) for the first 5 days after birth. Half of each group was then given gastric intubations of 20 mg/kg of 7,12-dimethylbenz(a)anthracene (DMBA) at 70, 77, and 84 days of age. The other half of each group was given sesame oil. Thus, this design yielded four experimental groups: oil + oil; 17 beta-estradiol + oil; oil + DMBA; and 17 beta-estradiol + DMBA. They were sacrificed at approximately 144 days of age (Experiment 1) or the day of palpable ovarian tumor detection or 360 days of age (Experiment 2). In Experiment 1, the total number of oocytes (follicles) per ovary in mice of the 17 beta-estradiol + oil group was maintained at the same level as mice of the oil + oil group. A significant reduction of oocytes, however, was observed in mice of the 17 beta-estradiol + DMBA group in comparison with mice of the oil + DMBA group (P less than 0.01), and neoplastic nodules of the granulosa cell type developed in the unilateral ovary in 10 of 17 mice of the 17 beta-estradiol + DMBA group. No tumors were detected in the mice of the other groups. The plasma levels of both follicle-stimulating and luteinizing hormones as determined by radioimmunoassay were significantly higher (P less than 0.01) in mice of the 17 beta-estradiol + oil group than in mice of the oil + oil group. In Experiment 2, more ovarian tumors of the granulosa cell type were detected before 360 days of age in mice of the 17 beta-estradiol + DMBA group (14 of 18) than in mice of the oil + DMBA group (5 of 15) (P less than 0.05). No tumors developed in mice of the other two groups. These results strongly indicate that an abnormal endocrine milieu caused by neonatal treatment with estrogen may induce a high frequency of transformation of some ovarian tissues and rapid growth of the ovarian tumors after DMBA treatment.

Influence of age on induction of mammary tumors by diethylstilbestrol in C3H/HeN mice with low murine mammary tumor virus titer.

C3H/HeN female mice with low murine mammary tumor virus titer (MTV-) were fed diets containing a targeted concentration of 640 ppb diethylstilbestrol [(DES) CAS: 56-53-1; 4,4'-(1,2-diethyl-1,2-ethenediyl)bis-phenol]. Mice were started on DES at 3, 5, 7, or 9 weeks of age. Some continued on the diet throughout the rest of their life-spans, whereas others were killed as soon as they had been fed DES for 2, 4, 6, 8, 10, or 12 weeks. Controls were also examined throughout the study. Among mice killed early, the only observation significantly influenced by age at the start of DES treatment was the presence or absence of corpora lutea (CL). DES did not prevent CL from appearing in mice started on DES at 7 or 9 weeks of age, but it did prevent their appearance in about 25% of the mice started at 5 weeks and in up to 75% of the mice started at 3 weeks of age. In the life-span-exposure groups, CL either disappeared or were never formed in 88% or more of the mice, regardless of age at the start of treatment. Neoplastic or presumptive preneoplastic lesions apparently influenced by DES in the life-span-treatment groups included ovarian tubular adenomas; granulosa cell tumors and luteomas; pituitary cystoid degeneration, hyperplasia, and adenomas; uterine adenocarcinomas and cervical adenosis; mesotheliomas; and mammary hyperplastic alveolar nodules (HANs) and adenocarcinomas. Luteoma and granulosa cell tumor incidences were reduced by DES, regardless of age at the start of treatment. Influence of age at the start of treatment was minimal or not apparent for mesotheliomas, uterine adenocarcinomas, or pituitary adenomas; however, pituitary cystoid degeneration and hyperplasia and cervical adenosis occurred in higher frequency and/or with shorter duration of DES exposure the earlier that treatment was started. A delay in the start of DES treatment was associated with a remarkable delay in HAN and mammary adenocarcinoma development. This was especially apparent in young mice (3-7 wk old) in which a 2-week delay in treatment resulted in a 20-week delay in HAN or tumor onset. Age at the start of treatment was a major factor in susceptibility of C3H/HeN-MTV- female mice to DES-induced mammary tumorigenesis.